The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Researchers have been developing methods for cloning mammalian animals over the past two decades. These reported methods typically include the steps of (1) isolating a pluripotent or totipotent cell; (2) inserting the cell or nucleus isolated from the cell into an enucleated oocyte (i.e., the oocyte's nucleus was previously extracted), and (3) allowing the embryo to mature in vivo.
The first successful nuclear transfer experiment using mammalian cells was reported in 1983, when pronuclei isolated from a murine (mouse) zygote were inserted into an enucleated oocyte and resulted in like offspring(s). See, e.g., McGrath & Solter, 1983, Science 220:1300-1302. Subsequently, other workers described the production of chimeric murine embryos (e.g., embryos that contain a subset of cells having significantly different nuclear DNA from other cells in the embryo) using murine primordial germ cells (PGCs). These cells are and can give rise to pluripotent cells (e.g., cells that can differentiate into other types of cells, and which may, but are not required to, differentiate into a grown animal). See, e.g., Matsui et al., 1992, Cell 70:841-847 and Resnick et al., 1992, Nature 359:550; Kato et al., 1994, Journal of Reproduction and Fertility Abstract Series, Society For the Study of Fertility, Annual Conference, Southampton, 13:38.
Progress has also been reported in the field of cloning ovine (sheep) animals (see, e.g., Willadsen, 1986, Nature 320:63-65; Campbell et al., 1996, Nature 380:64-66; PCT Publication WO 95/20042; Wilmut et al., 1997, Nature 385:810-813; PCT Publication WO 96/07732; PCT Publication WO 97/07668; and PCT publication WO 97/07669; and McCreath et al., 2000, Nature, 405:1066-1069), and bovine animals, (see, e.g., U.S. Pat. Nos. 4,994,384 and 5,057,420; Sims & First, 1993, Theriogenology 39:313; Keefer et al., 1994, Mol. Reprod. Dev. 38:264-268; Delhaise et al., 1995, Reprod. Fert. Develop. 7:1217-1219; Lavoir 1994, J. Reprod. Dev. 37:413-424; Stice et al., 1996, Biol. Reprod. 54: 100-110; and PCT application WO 95/10599 entitled “Embryonic Stem Cell-Like Cells”).
Researchers have also disclosed methods that resulted in cloned bovine animals (cattle). Bovines have been cloned using an embryonic cell derived from a 2-64 cell embryo as a nuclear donor. This bovine animal was reportedly cloned by utilizing nuclear transfer techniques set forth in U.S. Pat. Nos. 4,994,384 and 5,057,420. Others reported that cloned bovine embryos were formed where an inner cell mass cell of a blastocyst stage embryo was utilized as a nuclear donor in a nuclear transfer procedure (Sims & First, 1993, Theriogenology 39:313; Keefer et al., 1994, Mol. Reprod. Dev. 38:264-268; and U.S. Pat. No. 6,107,543); a PGC isolated from fetal tissue as a nuclear donor (Delhaise et al., 1995, Reprod. Fert. Develop. 7:1217-1219; Lavoir 1994, J. Reprod. Dev. 37:413-424; and PCT application WO 95/10599 entitled “Embryonic Stem Cell-Like Cells”); a proliferating somatic cell (U.S. Pat. No. 5,945,577); and a reprogrammed nonembryonic cell (U.S. Pat. No. 6,011,197)
Additionally, researchers have reported methods for obtaining cloned porcine animals and porcine chimeric animals, specifically, where a nuclear donor obtained from a 4-cell embryo is placed inside an enucleated zygote. See, e.g., Prather et al., 1989, Biology of Reproduction 41: 414-418; Piedrahita et al., 1998, Biology of Reproduction 58: 1321-1329; and WO 94/26884, “Embryonic Stem Cells for Making Chimeric and Transgenic Ungulates,” Wheeler, published Nov. 24, 1994. Also, researchers have reported nuclear transfer experiments using porcine nuclear donors and porcine oocytes. See., e.g., Nagashima et al., 1997, Mol. Reprod. Dev. 48: 339-343; Nagashima et al., 1992, J. Reprod. Dev. 38: 73-78; Prather et al., 1989, Biol. Reprod. 41: 414-419; Prather et al., 1990, Exp. Zool. 255: 355-358; Saito et al., 1992, Assis. Reprod. Tech. Andro. 259: 257-266; Terlouw et al., 1992, Theriogenology 37: 309, Pokajaeva et al., Nature 407, 86-90 (2000); Onishi et al., Science 289 1188-1190 (2000); and Betthauser et al., Nature Biotechnology 18: 1055-1059 (2000).
Researchers have also developed methods for generating transgenic cells, which may be applicable to the production of transgenic animals. Although several viral vectors, non-viral vectors, and other delivery systems have been developed for establishing transgenic cells, many of these technologies are constrained by multiple limitations. Specifically, these limitations include (1) the size of inserted DNA is limited to approximately 10 kilobases (kb); (2) integration of the DNA of interest cannot be specifically targeted into the cell's nuclear DNA; and (3) expression of a recombinant product from the DNA of interest cannot be well controlled. See, e.g., Mitani et al., 1993, Trends Biotech, 11: 162-166; U.S. Pat. No. 5,633,067, “Method of Producing a Transgenic Bovine or Transgenic Bovine Embryo,” DeBoer et al, issued May 27, 1997; U.S. Pat. No. 5,612,205, “Homologous Recombination in Mammalian Cells,” Kay et al., issued Mar. 18, 1997; and PCT publication WO 93/22432, “Method for Identifying Transgenic Pre-Implantation Embryos,” all of which are incorporated by reference herein in their entirety, including all figures, drawings, and tables.
Artificial chromosome technology is not constrained by the above-defined limitations. Moreover, researchers have discovered that artificial chromosomes can be replicated de novo. See, e.g., Kereso et al., 1996, Chromosome Research 4: 226-239, Holló et al., 1996, Chromosome Research 4: 240-247, U.S. Pat. No. 6,025,155, and U.S. Pat. No. 6,077,697.
Each reference used to provide background information in this section is hereby incorporated by reference in its entirety, including ant tables, figures, and claims.
Despite progress towards cloning mammals and establishing transgenic cells, there remains a great need in the art for materials and methods that enhance the efficiency for cloning transgenic animals. In particular, there remains a great need in the art to provide pluripotent and totipotent transgenic cells that can be utilized as nuclear donors. Furthermore, there remains a long felt need in the art for providing cell lines that are karyotypically stable and transgenic, which can be utilized in processes for cloning transgenic animals.